Bulk RNA-seq data analysis

Introduction

This tutorial is build on data from duodenal biopsies of active celiac disease, treated celiac disease, and control individuals, as described by Aarón Ramírez-Sánchez et al. (2024). Here you will learn the basics on how to import RNA-seq data, explore the data, build contrasts, perform differential expression analysis, and gene set enrichment analysis.

Initial set-up

Set working directory

In this step, you set your working directory, that is, the folder in your computer where you are going to save your scripts, data and results.

path <- '/Users/nvribeiro/Library/CloudStorage/OneDrive-UMCG/Desktop/PhD/Others/BMS-BigData-Course-2025' # you need to change this to your own path

setwd(path)

Load libraries

If you haven’t done, check the file “Preparation for data analysis – Big Data Course BMS 2025” and follow the instructions to install/update R, RStudio and the packages that you will used in the analysis. Then load the libraries.

library(dplyr)
library(tidyr)
library(ggplot2)
library(knitr)
library(stringr)
library(patchwork)
library(edgeR)
library(clusterProfiler)
library(enrichplot)
library(org.Hs.eg.db)
library(ReactomePA)
library(viridis)
library(ggrepel)
library(purrr)
library(biomaRt)
library(pheatmap)
library(RColorBrewer)
library(reshape2)
library(variancePartition)
library(forcats)

# Optional: define a default theme to be used in all plots
default_theme <- function(){
  theme_bw() +
    theme(panel.grid = element_blank(),
          panel.border = element_rect(linewidth = 1),
          axis.title = element_text(size = rel(1)),
          axis.title.y.left = element_text(margin = margin(r = 10, unit = 'pt')),
          axis.title.y.right = element_text(margin = margin(l = 10, unit = 'pt')),
          axis.title.x.bottom = element_text(margin = margin(t = 10, unit = 'pt')),
          plot.title = element_text(size = rel(1), hjust = 0.5, face = 'bold'),
          axis.text = element_text(size = rel(1)),
          axis.ticks.length = unit(5, 'pt'),
          strip.background = element_rect(fill = "#f5a9a9", linewidth = 0),
          strip.text = element_text(size = rel(1), color = "black")
    )
}

Load the dataset

Now we load the dataset that you have previously downloaded and saved in your “data” folder. This file contains the reads mapped to the gene annotation and thus contains the raw data with which we will work.

counts <- read.table("data/RNA_matrix.table", header=TRUE)
knitr::kable(head(counts))

	CON1	CON2	CON3	CON4	CON5	CON6	CON7	CON8	CON9	CON10	CON11	CON12	CON13	CON14	CON15	CON16	CON17	CON18	CON19	CON20	CON21	CON22	CON23	CON24	CON25	TCD1	UCD1	TCD2	TCD3	TCD4	TCD5	TCD6	TCD7	TCD8	TCD9	TCD10	TCD11	TCD12	TCD13	TCD14	TCD15	TCD16	TCD17	UCD2	UCD3	TCD18	TCD19	TCD20	UCD4	TCD21	UCD5	UCD6	TCD22	TCD23	TCD24	TCD25	UCD7	UCD8	UCD9	UCD10	UCD11	UCD12	UCD13	UCD14	UCD15	UCD16	TCD26	TCD27	UCD17	UCD18	UCD19	TCD28	UCD20	UCD21	UCD22	UCD23	UCD24	UCD25	UCD26	UCD27	UCD28	UCD29
ENSG00000000003	306	219	280	337	528	436	334	453	483	181	407	291	392	254	400	161	406	239	215	356	340	292	645	309	385	530	887	356	62	454	375	451	285	506	337	380	289	195	468	131	383	601	161	218	333	273	505	377	405	454	372	374	281	353	354	713	474	583	438	319	388	493	280	862	471	373	316	457	480	883	674	274	519	571	398	673	523	579	950	439	776	677
ENSG00000000005	0	0	0	0	0	0	8	0	0	1	0	0	0	0	0	0	2	0	0	2	3	2	0	0	6	0	13	0	0	0	0	0	0	0	8	0	0	0	0	0	0	10	0	0	11	0	0	0	7	6	0	0	0	0	0	0	0	15	5	1	33	0	0	0	0	0	0	1	2	44	2	0	4	2	19	0	3	0	41	54	22	10
ENSG00000000419	462	586	240	525	760	367	411	633	620	359	363	377	395	751	551	336	495	425	687	402	462	486	269	486	518	710	928	384	757	435	326	731	556	489	537	282	685	187	496	302	500	658	474	334	365	321	518	460	540	569	166	704	376	414	519	578	854	924	789	637	634	687	326	717	431	357	275	238	493	758	580	618	574	549	475	465	398	516	1155	775	666	516
ENSG00000000457	327	318	374	473	429	305	409	243	248	268	283	237	279	223	170	310	354	309	200	305	237	361	187	298	431	369	355	375	261	286	142	529	196	438	424	210	322	121	351	243	226	302	256	244	185	149	364	312	260	367	471	70	377	274	181	551	467	314	322	311	348	258	147	439	368	192	260	188	276	375	297	220	223	235	177	295	235	471	530	210	235	304
ENSG00000000460	58	41	125	134	21	69	33	34	22	33	21	45	40	83	41	33	65	59	105	61	53	65	68	54	38	76	232	120	203	100	179	71	86	37	76	33	43	33	63	52	101	70	10	35	53	21	7	41	93	64	8	150	111	52	55	51	119	169	184	61	239	65	61	119	76	57	49	40	95	114	78	6	99	81	56	52	110	109	145	21	119	37
ENSG00000000938	827	466	491	281	458	320	467	1111	182	367	375	391	319	748	454	629	198	315	448	371	237	326	225	372	511	426	33	375	119	66	304	345	516	302	503	262	522	194	226	384	165	114	651	823	272	137	95	173	131	489	33	299	524	192	151	796	461	465	101	275	494	261	278	199	438	130	74	268	438	71	144	366	221	180	138	90	172	608	394	355	57	86

We now have a dataframe where each row is a gene and each column is sample.

Preparing the metadata

Differential expression analysis is the comparison of gene expression between one or more conditions while correcting for unwanted sources of variation. This information is usually stored in a metadata file, where you have many details about each sample such as condition, sex, age, etc. Here we load the metadata for this dataset.

metadata <- read.csv('data/metadata_oslo_biopsies.csv', header=TRUE)
rownames(metadata) <- metadata$sample

# Visualize the first rows
knitr::kable(head(metadata))

	sample	condition	marsh.score	sex	age	H..pylori	PPI	cell.counts	batch.of.RNA.isolation	batch.of.sequencing	RNA.concentration	RIN.score	total.reads	GC.content	crypt.ratio..APOA4.KI67.	group
CON1	CON1	CTRL	n.m.	M	37	NA	Yes	1500000	2	A	2.28	7.3	2402861	47.5	1.487207	Mild inflammation
CON2	CON2	CTRL	n.m.	M	26	NA	Yes	1500000	5	A	8.00	7.9	2644867	46.0	1.666809	Non-inflamed
CON3	CON3	CTRL	n.m.	F	27	NA	No	1267000	5	A	4.09	8.1	2548105	47.0	1.492771	Mild inflammation
CON4	CON4	CTRL	n.m.	F	38	NA	Yes	1620000	4	A	5.81	7.7	2812289	47.0	1.697659	Non-inflamed
CON5	CON5	CTRL	n.m.	M	86	NA	Yes	1800000	3	A	12.28	8.2	2843597	46.0	1.631452	Non-inflamed
CON6	CON6	CTRL	n.m.	M	65	NA	No	1800000	3	A	4.13	9.0	1730893	46.0	1.447205	Mild inflammation

# Make sure that the colnames of your count table are matching with the row names of your colData
# Both of the comparisons below need to return TRUE
all(rownames(metadata) %in% colnames(counts)) # check if all samples in the metadata are present in the counts

[1] TRUE

all(rownames(metadata) == colnames(counts)) # check if they are in the same order

[1] FALSE

# In this case all samples are in counts and metadata, but they are not in the same order.
# To put them in the same order we run
counts <- counts[, rownames(metadata)]
all(rownames(metadata) == colnames(counts)) # now this is TRUE

[1] TRUE

Quality control and exploratory analysis

Now we are going to create the edgeR object that will contain all the information about our experiment. We need to give the counts table, the matching metadata and a optional grouping factor (which we are not using in this case).

y <- DGEList(counts = counts,
            samples = metadata,
            genes = rownames(counts))

Remove poorly expressed genes

As we sequenced this data set very deeply, a lot of noise is also captured, which is most prevalent in lowly expressed genes. To reduce the size of our data set and to make the differential expression analysis more robust by including only well-expressed genes that are more likely to be biologically informative, we will remove poorly expressed genes.

First we visualize the counts in a histogram, this will give us an overview of the expression and we can try to decide a cutoff based on this graph. Here we are plotting the distribution of counts as log10 count-per-million (CPM).

# Histogram of log(CPM)
cpm <- cpm(y, log = TRUE)
hist(cpm)

# With the following lines we add arbitrary cut-offs to histogram
# Cutoff of 10
abline(v = log10(10), col = "blue",lwd = 2)
# cutoff of 100
abline(v = log10(100), col = "purple",lwd = 2)
# cutoff of 500
abline(v = log10(500), col = "red",lwd = 2)
# cutoff of 1000
abline(v = log10(1000), col = "orange",lwd = 2)

You will notice a bi-modal distribution, in which a large chunk of genes is barely expressed, and a smaller chuck is expressed much more. We see that the bump with highly expressed genes occurs after the purple line (100 counts), so we can use that as a cut-off or, if you want to focus only on the really highly expressed genes, 500 or 1000 counts. Here I chose 500.

The function we will use to filter, filterByExpr, allows us to specify some parameters as the minimum counts required, if you want to filter per group, or use a formula.

keep <- filterByExpr(y,
                    group = y$samples$condition, # considers the group when filtering - optional
                    min.count = 500) # the number you decided based on the histogram above

y_filtered <- y[keep, , keep.lib.sizes=FALSE]

print(paste0("Number of genes before filtering: ", dim(y)[1]))

[1] "Number of genes before filtering: 53042"

print(paste0("Number of genes after filtering: ", dim(y_filtered)[1]))

[1] "Number of genes after filtering: 6809"

We now reduced our dataset from 53,042 genes to 6,809. This will help speed up the calculations and only focus on genes that are expressed at high levels and probably more biologically relevant.

Principal component analysis (PCA)

The PCA can help assess which samples are more closely related to each other, and how much variation is found between samples. We can also use PCA to check if other variables in our metadata are unwanted sources of variation that should be included in the model. To calculate the PCA, we use the logCPM counts.

Tip

If you are not familiar with PCA, watch this video: https://www.youtube.com/watch?v=HMOI_lkzW08

# First define a function to make easier to plot the PCs. This function makes a PCA plot for the any PCs specified in "coord_1" and "coord_2" and will group the points (color) by a metadata column specified in "color_by". "coldata" is your metadata (stored in the "samples" part of the edgeR object)
plotPC <- function(pca_obj, coldata, coord_1, coord_2, color_by) {

  var_explained <- pca$sdev^2 / sum(pca$sdev^2)
  var_explained_df <- tibble(PC = paste0('PC', seq_along(var_explained)),
                             Var_Explained = var_explained)
  
  df <- as.data.frame(cbind(coldata, pca$x))
  
  x_label <- paste0(coord_1, ' (', round(var_explained_df$Var_Explained[var_explained_df$PC == coord_1]*100, digits = 2), '%)')
  y_label <- paste0(coord_2, ' (', round(var_explained_df$Var_Explained[var_explained_df$PC == coord_2]*100, digits = 2), '%)')
  
  p <- ggplot(df, aes(x = !!sym(coord_1), y = !!sym(coord_2), color = as.factor(!!sym(color_by)))) +
    geom_point(size = 3) +
    scale_color_viridis_d() +
    labs(x = x_label, y = y_label) +
    default_theme()
  
  return(p)
  
}

# The next function we define is to create a scree plot
# Makes a Scree plot based on the PCA results, if n_pcs = NULL, plots all available PCs
plotScree <- function(pca, n_pcs = NULL){
  
  if (is.null(n_pcs)){
    n_pcs <- ncol(pca$x)
  }
  
  # Extract variance explained
  var_explained <- pca$sdev^2 / sum(pca$sdev^2)
  cumulative_var <- cumsum(var_explained)
  
  # Create a data frame for plotting
  plot_data <- data.frame(
    PC = seq_along(var_explained),
    Variance_Explained = var_explained,
    Cumulative_Explained = cumulative_var
  )
  
  plot_data <- plot_data[1:n_pcs,]
  
  # Plot
  ggplot(plot_data, aes(x = factor(PC))) +
    geom_bar(aes(y = Variance_Explained), stat = "identity", fill = "#22A884FF") +
    geom_line(aes(y = Cumulative_Explained, group = 1), color = "#2A788EFF", linewidth = 1) +
    geom_point(aes(y = Cumulative_Explained), color = "#2A788EFF", size = 2) +
    scale_y_continuous(
      name = "Fraction of Variance Explained",
      sec.axis = sec_axis(~., name = "Cumulative Explained Variance"),
      expand = c(0,0)
    ) +
    labs(
      title = "Scree Plot",
      x = "Principal Component",
      y = "Fraction of Variance"
    ) +
    default_theme()
  
}

# Now calculate the PCs and plot
cpm <- cpm(y_filtered, log = TRUE)
pca <- prcomp(t(cpm), scale. = TRUE)

First we can see how much variation is explained by the PCs using a scree plot. For example, we can see that the first 3 PCs already explain almost 60% of the variance, and after each PCs doesn’t add too much to the variance. That means that if we see a variable having an effect in the first 3 PCs, it is probably a good idea to correct for that during the testing.

plotScree(pca, n_pcs = 20)

Here you can see that the samples from controls (CTRL) and treated celiac disease (TCD) tend to cluster together and separated from the untreated celiac disease (UCD).

# Plot the first 2 PCs colored by the condition
plotPC(pca,
      coldata = y_filtered$samples,
      coord_1 = 'PC1',
      coord_2 = 'PC2',
      color_by = 'condition')

Here you can see that the samples from controls (CTRL) and treated celiac disease (TCD) tend to cluster together and separated from the untreated celiac disease (UCD). You can plot any other PCs and grouping variable you want to explore how that variable influences the data, for example, batch of sequencing that seems to be a significant source of variation:

plotPC(pca,
      coldata = y_filtered$samples,
      coord_1 = 'PC1',
      coord_2 = 'PC2',
      color_by = 'batch.of.sequencing')

Normalization of read counts

In order to properly perform certain downstream analysis, such as principal component analysis (PCA), or clustering, we need to properly normalize the counts. This step adjusts the raw counts to account for technical variations that can mask true biological differences. These factors are things like sequencing depth, library sizes, gene length, and GC content. In edgeR, we normalize the counts using the trimmed mean M-values (TMM) method with the function normLibSizes

y_filtered <- calcNormFactors(y_filtered)

Variance partition analysis (optional)

Alternatively to the PCA, you can use a “Variance Partition analysis” to quantify and interpret the sources of biological and technical variation in your data. This uses the package variancePartition. This uses the TMM normalized counts and a formula with the variables from your metadata you want to check (more on models and formulas will be explained in future steps). You can investigate any variables you want at once here, instead of one by one as you would do in the PCA.

# First we assess correlation between all pairs of variables
form <- ~ condition + sex + age + batch.of.RNA.isolation + batch.of.sequencing + RNA.concentration + RIN.score + lib.size + cell.counts + H..pylori + marsh.score
C <- canCorPairs(form, y_filtered$samples)

Warning in canCorPairs(form, y_filtered$samples): Regression model may be problematic.
High colinearity between variables:
  condition and marsh.score

plotCorrMatrix(C)

We see from this plot that GC content, RIN score, batch of isolation and batch of sequencing are all strongly correlated, so we don’t want to include all of them in our model. Because batch of sequencing was also important in the PCA, let’s use just that one.

Now we are going to fit a model in the gene expression data to check how much each variable contributes to the variation, you can choose which variables to include based on the previous plot.

design <- model.matrix(~condition, y_filtered$samples)
v <- voom(y_filtered, design)

# We need to scale some variables so they are all in the same scale and comparable
y_filtered$samples$age <- scale(y_filtered$samples$age)
y_filtered$samples$lib.size_scaled <- scale(y_filtered$samples$lib.size)
y_filtered$samples$cell.counts_scaled <- scale(y_filtered$samples$cell.counts)

# Categorical variables need to be specified with (1|variable)
form <- ~ (1|condition) + (1|sex) + (1|batch.of.sequencing) + (1|H..pylori) + (1|marsh.score) + age + RNA.concentration + lib.size_scaled + cell.counts_scaled
varPart <- fitExtractVarPartModel(v, form, y_filtered$samples)

Warning: 
Variables contain NA's: H..pylori, age 
Samples with missing data will be dropped.

Warning in .fitExtractVarPartModel(exprObj, formula, data, REML = REML, : Model failed for 2 responses.
  See errors with attr(., 'errors')

plotVarPart(varPart)

In this plot, we can see how much each variable contributes to the variance in the data (the model is fit for every gene, that’s why you see a distribution). Quite some variance is explained by condition (and therefore also Marsh score, a measure of inflammation in CeD), as you would expect. Batch of sequencing, sex, age, and RNA concentration are all also some significant sources of variation that we could include in the model. However, we see that there is still a lot of variance left in the residuals - that means variance that is not being captured by any of the other variables in the model. This can happen for many reasons that we won’t go into details here, but keep in mind that ideally you want a model where most of variance is explained by the variables in the model and you don’t have much left in the residuals.

Differential expression analysis

The definition of a differential expressed gene (DEG) usually depends on whether or not a particular gene is significantly (p-value = 0.05, after multiple testing correction) over or under expressed when compared to another class (can be different treatment, control, tissue etc,). Nevertheless, we can be a bit more strict in this definition, we could require either a lower p-value (such as 0.01, or 0.001), and require a certain difference of expression between the two conditions, this difference between two conditions can be interpreted as the log2 fold change: \(log2FC = log2 (mean expression condition 1 /mean expression condition 2)\)

Now that we have looked at the data and seen what things may affect the expression of genes in there, we can start making a design matrix to do differential expression analysis. A design matrix is a table that represents your experimental setup in a mathematical form. It tells a statistical model which samples belong to which experimental groups, such as “diseased” vs. “healthy” and also how other variables (such as sex, age, etc.) affect the sample. For a more detailed explanation on how to design matrices for many types of experimental designs, check this guide.

Building a design matrix

The first thing in you want to include in your design is of course the variable you are more interested on, for example here we want to find the genes that are differentially expressed because of condition . Next, you want to include your covariates: the variables that we identified above that influence the data but are not the object of our investigation, so we correct for them. You can write the formula for the design matrix in two ways - with or without intercept - and to do that we use the function model.matrix and a formula, which always starts with a ~ followed by your variables, for example ~ condition + sex + age.

Model with intercept

Let’s use a simple example where we are modeling the effect of group (disease or control), the design matrix can be created with model.matrix(~group). This creates a model with intercept. The intercept is the reference level for group, in this case, “control”. In this case, gene expression is modeled as Y ~ a + Xb, where Y is the expression, a is the intercept, X is 0 (control) or 1 (disease), and b represents how much the expression changes from the reference group. . When X = 0, Y = a, the expression for control. When X = 1, Y = a + b, the expression for disease. Below is a visual representation of how to interpret this.

Model without intercept

A design matrix without intercept is coded as model.matrix(~0+group). This model is essentially the same as before, however now you have a term associated with group control and a term associated with group disease: Y ~ aX1 + bX2, where X1 is 1 for control and 0 for disease and X2 is 1 for disease and 0 for control.

The result you get from both models is the same, but for experimental designs with more than 2 groups (like our data) and other complex designs, is easier to work with the model without intercept. That is because having one term for each group allows you to easily define the contrasts for exactly what you want to test and find the DEGs for.

Defining a contrast matrix

Now that we have a model for the gene expression, we want to find the difference between the mean expression of the groups of interest. The difference in parameter estimates can be calculated using a contrast matrix via the makeContrast function. To specify your comparison of interest, use the columns from the design matrix, for example:

## DO NOT RUN ##
design <- model.matrix(0+group)
makeContrasts(groupSICK - groupHEALTHY, levels = colnames(design))

This will create the contrast to find the DEGs in sick vs healthy that we will use in the following steps. Note that this is only applicable when creating a design matrix without intercept. Back to our data, this is how we will define our design matrix and contrasts, including the covariates:

# Here it is also important that the categorial variables are factors and the numerical variables are on the same scale
y_filtered$samples$condition <- factor(y_filtered$samples$condition, levels = c('CTRL', 'TCD', 'UCD'))
y_filtered$samples$batch.of.sequencing <- factor(y_filtered$samples$batch.of.sequencing)
y_filtered$samples$sex <- factor(y_filtered$samples$sex)
y_filtered$samples$RNA.concentration <- scale(y_filtered$samples$RNA.concentration)

# The parameter data in model.matrix() is your metadata (y$samples)
design <- model.matrix(~ 0 + condition + batch.of.sequencing + sex + RNA.concentration, data = y_filtered$samples)
head(design)

     conditionCTRL conditionTCD conditionUCD batch.of.sequencingB sexM
CON1             1            0            0                    0    1
CON2             1            0            0                    0    1
CON3             1            0            0                    0    0
CON4             1            0            0                    0    0
CON5             1            0            0                    0    1
CON6             1            0            0                    0    1
     RNA.concentration
CON1        -0.7021056
CON2        -0.4025453
CON3        -0.6073147
CON4        -0.5172371
CON5        -0.1783988
CON6        -0.6052199

Now we specify the comparisons we want to make. There are many possible combinations we can investigate here, for example: untreated celiac disease vs control, untreated celiac disease vs treated celiac disease, and so on… When working on your own analysis think about the biological questions you want to answer and define the contrasts accordingly.

contrasts_matrix <- makeContrasts(
    UCDvsCTRL = conditionUCD - conditionCTRL,
    TCDvsCTRL = conditionTCD - conditionCTRL,
    UCDvsTCD = conditionUCD - conditionTCD,
    levels = colnames(design)
)
head(contrasts_matrix)

                      Contrasts
Levels                 UCDvsCTRL TCDvsCTRL UCDvsTCD
  conditionCTRL               -1        -1        0
  conditionTCD                 0         1       -1
  conditionUCD                 1         0        1
  batch.of.sequencingB         0         0        0
  sexM                         0         0        0
  RNA.concentration            0         0        0

Now that we have our models and contrasts, we are finally ready to find the DEGs.

Fit the model and find DEGs

In the following steps we perform the necessary steps to fit the model and extract the DEGs for the comparisons we want. The function voom transforms the raw counts and stabilized variance, so the data can be modeled with limma’s linear model.

v <- voom(y_filtered, design, plot=TRUE) # change to FALSE if you don't want the plot

The plot generated by voom is a diagnostic tool that visualizes the relationship between a gene’s average expression and its variance. The plot above is a example of a good plot: it has a “smooth”, downward-sloping trend line, showing that voom has captured the mean-variance relationship. A bad plot usually has “peaks” and looks irregular, suggesting there is a problem with the data, such as bad filtering of lowly expressed genes or technical issues.

Now we fit the model to the voom-transformed data and extract the results.

# Fit the model
fit <- lmFit(v, design)
fit2 <- contrasts.fit(fit, contrasts_matrix[, "UCDvsCTRL"]) # here is where you specify the contrast you want the results for
fit2 <- eBayes(fit2)

# Get the table with results
res <- topTable(fit2, sort.by = "P", n = Inf) # this returns the table with the results for all the genes, ordered by p-value
knitr::kable(head(res))

	genes	logFC	AveExpr	t	P.Value	adj.P.Val	B
ENSG00000117228	ENSG00000117228	1.9855499	6.367871	13.50997	0	0	40.64992
ENSG00000092010	ENSG00000092010	0.7266291	7.670674	12.78049	0	0	37.62139
ENSG00000204264	ENSG00000204264	1.1348134	6.870195	12.75139	0	0	37.49876
ENSG00000148773	ENSG00000148773	2.7271550	6.506489	12.70288	0	0	37.28963
ENSG00000182054	ENSG00000182054	1.7045698	6.584646	12.47710	0	0	36.33895
ENSG00000182481	ENSG00000182481	2.0288446	4.891909	12.29831	0	0	35.47388

These are the results for just untreated celiac disease vs control. Instead of running the code above multiple times for each comparison, we can do this iterative for all comparisons.

res_all <- list()
for (x in colnames(contrasts_matrix)) {
  
  contr <- contrasts_matrix[, x]
  
  fit2 <- contrasts.fit(fit, contr)
  fit2 <- eBayes(fit2)
  res <- topTable(fit2, sort.by = "P", n = Inf)
  res$Contrast <- x
  res_all <- append(res_all, list(res))
    
}
results <- list_rbind(res_all)
knitr::kable(head(results))

	genes	logFC	AveExpr	t	P.Value	adj.P.Val	B	Contrast
ENSG00000117228…1	ENSG00000117228	1.9855499	6.367871	13.50997	0	0	40.64992	UCDvsCTRL
ENSG00000092010…2	ENSG00000092010	0.7266291	7.670674	12.78049	0	0	37.62139	UCDvsCTRL
ENSG00000204264…3	ENSG00000204264	1.1348134	6.870195	12.75139	0	0	37.49876	UCDvsCTRL
ENSG00000148773…4	ENSG00000148773	2.7271550	6.506489	12.70288	0	0	37.28963	UCDvsCTRL
ENSG00000182054…5	ENSG00000182054	1.7045698	6.584646	12.47710	0	0	36.33895	UCDvsCTRL
ENSG00000182481…6	ENSG00000182481	2.0288446	4.891909	12.29831	0	0	35.47388	UCDvsCTRL

This list have the genes with their Ensembl ID, to help with interpretation we will add the gene symbol and entrezid annotation before we continue.

mart <- useEnsembl(biomart = "ensembl", dataset = "hsapiens_gene_ensembl")
genemap <- getBM(attributes = c("ensembl_gene_id", "hgnc_symbol", "entrezgene_id"),
                 filters = "ensembl_gene_id",
                 values = unique(results$genes),
                 mart = mart)
                        
results <- left_join(results, genemap, by = join_by("genes" == "ensembl_gene_id"), relationship = 'many-to-many')
knitr::kable(head(results))

genes	logFC	AveExpr	t	P.Value	adj.P.Val	B	Contrast	hgnc_symbol	entrezgene_id
ENSG00000117228	1.9855499	6.367871	13.50997	0	0	40.64992	UCDvsCTRL	GBP1	2633
ENSG00000092010	0.7266291	7.670674	12.78049	0	0	37.62139	UCDvsCTRL	PSME1	5720
ENSG00000204264	1.1348134	6.870195	12.75139	0	0	37.49876	UCDvsCTRL	PSMB8	5696
ENSG00000148773	2.7271550	6.506489	12.70288	0	0	37.28963	UCDvsCTRL	MKI67	4288
ENSG00000182054	1.7045698	6.584646	12.47710	0	0	36.33895	UCDvsCTRL	IDH2	3418
ENSG00000182481	2.0288446	4.891909	12.29831	0	0	35.47388	UCDvsCTRL	KPNA2	3838

We can also create a dataframe containing only the significant results. Here we are considering significant the genes with absolute log2FC > 1 and adjusted p-value < 0.05.

results_significant <- results |>
    dplyr::filter(abs(logFC > 1) & adj.P.Val < 0.05)

# Save the results
# Specify the path to the folder you want to save the results
folder_path <- "results"

# Check if the folder exists
if (!dir.exists(folder_path)) {
  # If it doesn't exist, create it
  dir.create(folder_path)
  cat(paste0("Folder '", folder_path, "' created successfully!\n"))
} else {
  cat(paste0("Folder '", folder_path, "' already exists.\n"))
}

Folder 'results' already exists.

write.csv(results, paste0(folder_path, "/DEG_results_biopsies_oslo.csv"), row.names=F)
write.csv(results_significant, paste0(folder_path, "/DEG_results_biopsies_oslo_significant_only.csv"), row.names=F)

Visualization

Volcano plot

Now we can visualize the DEGs in a volcano plot, which plots the -log10(padj) against the fold change of the genes. This allows for easy identification of data points that show both significant changes and large magnitudes of change.

# Define a function to make a volcano plot
# Makes a volcano plot, highlighting the genes above the specified log2FC threshold and labels the top n genes (based on padj)
VolcanoPlot <- function(res, threshold = 0.5, p_cutoff = 0.05, n_labels = 15, title = '') {
    # Prepare the data
  res_plot <- res |>
    mutate(DE = case_when(
      adj.P.Val < p_cutoff & logFC >= threshold ~ 'upregulated',
      adj.P.Val < p_cutoff & logFC <= -threshold ~ 'downregulated',
      adj.P.Val >= p_cutoff | abs(logFC) < threshold ~ 'not DE'
    ))
  
  # Add label to the top n genes (ranked by FC)
  n <- n_labels
  
  top_genes <- res_plot |> dplyr::filter(DE != 'not DE') |> 
    group_by(sign(logFC)) |> 
    top_n(n_labels, -log10(adj.P.Val)) |> 
    pull(hgnc_symbol)
  res_plot$genelabel <- ''
  res_plot$genelabel[res_plot$hgnc_symbol %in% top_genes] <- res_plot$hgnc_symbol[res_plot$hgnc_symbol %in% top_genes]
  
  color_map <- c("not DE" = "gray", "downregulated" = "#2166ac", "upregulated" = "#b2182b")
  
  ggplot(res_plot, aes(x = logFC, y = -log10(adj.P.Val))) +
    geom_point(aes(colour = DE), size = 1.5) +
    geom_text_repel(aes(label = genelabel), min.segment.length = 0.25, force = 10) +
    geom_hline(yintercept = -log10(p_cutoff), colour = '#696969', linetype = 'dashed') +
    geom_vline(xintercept = -threshold, colour = '#696969', linetype = 'dashed') +
    geom_vline(xintercept = threshold, colour = '#696969', linetype = 'dashed') +
    scale_color_manual('', values = color_map) +
    default_theme() +
    theme(legend.text = element_text(size = rel(1.25))) +
    labs(title = title,
         x = 'log2 fold change',
         y = '-log10 adjusted p-value')
  
}

Like before, we do this in a iterative way to make the volcano plots for all comparisons at the same time. You can change the log2FC threshold or the p_cutoff based on your cut-offs for a gene to be considered DE.

volcanos <- list()
for(c in unique(results$Contrast)) {
    data <- dplyr::filter(results, Contrast == c)
    p <- VolcanoPlot(data, threshold = 0.5, p_cutoff = 0.05, n_labels = 15, title = c)
    volcanos <- append(volcanos, list(p))
}
wrap_plots(volcanos, guides = 'collect')

Heatmap

We can also visualize the DEGs in a heatmap. Heatmaps are color coded, graphical representations of a matrix. The rows and columns of this matrix can be arranged in a certain way to showcase the similarities between columns and rows. This arrangement of columns and rows can be used an unsupervised approach such as hierarchical clustering, which can be useful to find patterns in the data, such as cluster of genes that are down/up-regulated with similar magnitude. It is also useful to visualize the gene expression per sample. To create this plot we use the expression data as logCPM from the significant genes.

# Choose a contrast to visualize
contrast <- 'UCDvsCTRL'

# Get the DEGs for this contrast
de_genes <- results_significant$genes[results_significant$Contrast == contrast]

# Get the logCPM counts for these genes
cpm_de <- cpm(y_filtered, log = TRUE)
cpm_de <- cpm_de[de_genes,]

# Calculates z-score
z <- t(scale(t(cpm_de)))

# Create annotation dataframe for the heatmap - this can be whatever you want to show about the samples (columns)
annotation_col <- y_filtered$samples[colnames(z), c('condition', 'marsh.score')]
rownames(annotation_col) <- y_filtered$samples[colnames(z), 'sample']
ann_colors <- list(
    condition = c('CTRL' = '#29d3d6',
                 'TCD' = '#29d62e',
                 'UCD' = '#910a0a'),
    marsh.score = c('n.m.' = '#FEE5D9',
                   '0' = '#FCAE91',
                   '1' = '#FB6A4A',
                   '2' = '#DE2D26',
                   '3' = '#A50F15'))

# Make the heatmap with values scaled by row (genes), so you can compare between samples (columns)
pheatmap(z, cluster_rows = T, show_rownames = F, show_colnames = F, border_color = NA,
        color = colorRampPalette(rev(brewer.pal(n = 7, name = "RdBu")))(100),
        annotation_col = annotation_col,
        annotation_colors = ann_colors,
        treeheight_col = FALSE)

Data points

In some cases it’s also useful to visualize the expression values for certain genes across all samples. Lets check for the top 10 DEGs between untreated celiacs and controls. Remember, in order to properly compare expression values across samples we need to use the normalized levels (in this case logCPM).

# Select the top 10 genes (based on adj p-val) for UCDvsCTRL
top10_genes <- results_significant |>
    dplyr::filter(Contrast == 'UCDvsCTRL') |>
    top_n(-10, adj.P.Val)

# Get the cpm counts
top10_cpm <- cpm(y_filtered, log=TRUE)
top10_cpm <- as.data.frame(cpm[top10_genes$genes,])
top10_cpm$symbol <- top10_genes$hgnc_symbol # add the gene symbol

# Reshape the data so we can plot
data_plot <- melt(top10_cpm)

Using symbol as id variables

# Add sample information (condition)
data_plot <- left_join(data_plot, y_filtered$samples[, c('sample', 'condition')], by = join_by("variable" == "sample"))

top10_Plot <- ggplot(data_plot, aes(x = condition, y = value))+
                geom_jitter(alpha=0.8)+
                geom_boxplot(alpha=0.6)+
                facet_grid(~symbol, scale="free")+
                ylab("logCPM")+
                xlab("")+
                default_theme()+
                theme(axis.text.x = element_text(angle=45, hjust = 1))
top10_Plot

Now we have our DEGs and many ways to visualize them. But what do they do? How are they biologically relevant?

Most of the times we have hundreds or thousands of DEGs, so it might be hard to look at them individually and check their function. To deal with that, we can do a pathway analysis to find if the DEGs are involved in common pathways that are enriched in the results.

Pathway analysis

To find if our DEGs are enriched for a particular biological function we can use a Over Representation Analysis (ORA) or a Gene Set Enrichment Analysis (GSEA). ORA is a method to find if known biological functions are over-represented in a given list of genes (e.g. DEGs), comparing to a background of all genes measures. A limitation of this method is that it doesn’t take into account how much the expression of a gene changes (i.e. the log2 fold-change). GSEA, on the other hand, uses the log2 fold-change information to rank all genes and calculate an enrichment score that reflects how clustered the genes from a particular set are at the top (upregulated) or bottom (downregulated) of the ranked list. There are many tools and databases with gene biological function that you can use, in this tutorial we will use the package clusterProfiler and the databases gene ontology, KEGG, and Reactome.

Prepare the data

Load the results from the DE analysis. Here let’s use all genes that are significant (adj. p-value < 0.05) but without filtering for the fold-change, so we can see how the results from ORA and GSEA will differ from each other. In your own analysis, you can set a fold-change cut-off as well if necessary. Besides, we need the Entrezid for most of the methods so we will filter out genes that don’t have one.

# Load the results and keep only the significant genes and remove genes without entrezid
results <- read.csv('results/DEG_results_biopsies_oslo.csv')
results_sig <- results |> 
    filter(adj.P.Val < 0.05 & !is.na(entrezgene_id))

Over Representation Analysis

The function compareCluster allows us to run ORA for the three comparisons and two directions (up or downregulated) at the same time. The function needs a formula specifying how you want to run the comparison. To tell we want the analysis per contrast and for each direction in that contrast we use the formula entrezgene_id ~ Contrast + direction. In this analysis, you can also specify a “universe”: a list of all genes that were tested in your analysis, to be used as a background. (This block of code might take a few minutes to run.)

# Create a column with direction of DE
results_sig$direction <- if_else(results_sig$logFC > 0, "upregulated", "downregulated")

# Get the universe - all genes tested
universe <- results |> filter(!is.na(entrezgene_id)) |> pull(entrezgene_id)

# Run ORA using gene ontology biological process
ora_go <- compareCluster(entrezgene_id ~ Contrast + direction,
                        fun = 'enrichGO',
                        universe = universe,
                        ont = "BP",
                        OrgDb = org.Hs.eg.db,
                        data = results_sig,
                        pvalueCutoff = 0.05,
                        readable = TRUE)

# Run ORA using KEGG Pathway
ora_kegg <- compareCluster(entrezgene_id ~ Contrast + direction,
                        fun = 'enrichKEGG',
                        universe = universe,
                        organism = "hsa",
                        data = results_sig,
                        pvalueCutoff = 0.05)

# Run ORA using Reactome
ora_reactome <- compareCluster(entrezgene_id ~ Contrast + direction,
                        fun = 'enrichPathway',
                        universe = universe,
                        organism = "human",
                        data = results_sig,
                        pvalueCutoff = 0.05,
                        readable = TRUE)

These objects contain a lot of information about the test and results. You can access the table with results with @compareClusterResult, for example:

knitr::kable(head(ora_go@compareClusterResult))

Cluster	Contrast	direction	ID	Description	GeneRatio	BgRatio	RichFactor	FoldEnrichment	zScore	pvalue	p.adjust	qvalue	geneID	Count
UCDvsCTRL.downregulated	UCDvsCTRL	downregulated	GO:0006631	fatty acid metabolic process	106/1700	405/18986	0.2617284	2.923044	12.267746	0	0	0	PCK1/ACAT1/ACADM/CYP3A4/CYP2J2/LPIN2/LIPA/ABHD6/CD36/EPHX1/CYP2C9/ASAH2/ABHD2/HACL1/ACSF2/PRKAB2/CYP4F2/ECHDC2/GPX4/RGN/APPL2/CYP4V2/ACOX1/APOA4/ACBD4/NAAA/PTGR1/APOC3/HADHB/ACAA1/MLXIPL/PDK4/DEGS1/NR1H3/CYP2D6/CYP4F12/BDH2/CROT/CRAT/CRYL1/HPGD/PCK2/PDK2/ACBD5/ACSS1/ABCD4/ACSL5/PHYH/GSTM2/PPARA/HACD2/KAT2B/CES2/SLC27A4/ACADSB/GGT1/ETFDH/FABP2/SIRT2/CYP2S1/CYP2C18/ACOX2/ACAA2/ABHD5/UGT1A10/ABHD12/CYP2C19/ASAH1/CYP4F3/ADH4/SGPL1/INSIG2/ACADVL/CEACAM1/WDTC1/PPARG/LPGAT1/PRKAG2/SCP2/UBR4/DCAF5/ABCD1/ALOX5/SESN2/HPGDS/PDK3/PTGS1/AIG1/ALDH3A2/ALOX5AP/SCAP/ACOT11/CBR1/HADHA/TNFRSF1A/LPIN3/NR1H2/ABCD3/PEX5/ACSL1/CPT2/SREBF1/MGLL/AUH/ACADS/MGST3	106
UCDvsCTRL.downregulated	UCDvsCTRL	downregulated	GO:0046486	glycerolipid metabolic process	96/1700	385/18986	0.2493506	2.784807	11.095220	0	0	0	PCK1/APOA1/LPIN2/LIPA/APOB/ABHD6/ABHD2/SERINC1/SORL1/SCARB1/RGN/APOA4/NAAA/DGAT2/DGKA/ENPP2/APOC3/GK/DGAT1/LPCAT3/DDHD2/NR1H3/AGPAT3/PIP4K2A/PCK2/PDGFA/G6PC1/PLCH2/PIP4P1/MTMR11/MTMR4/MTMR3/TTC7A/GPAM/CIDEC/GPD1/INPP5K/GPAT3/ABHD5/CDIPT/PIK3R5/ABHD12/PI4K2B/SLC44A4/APOBEC1/MBOAT7/INSIG2/GK5/PNPLA2/LPGAT1/PIKFYVE/CAT/AVIL/PI4KA/PAFAH1B1/MTTP/PGS1/PIP5K1C/CDK8/BMX/PI4KB/PLCG2/MOGAT2/AADAC/HYCC2/MECP2/GPCPD1/CHKA/CEPT1/TAFAZZIN/PIP5K1B/ABHD4/MTMR12/HADHA/PGAP3/MOGAT3/LPIN3/PCYT1A/INPP5J/IPMK/PISD/NR1H2/PIK3CG/ACSL1/PNPLA6/SREBF1/PRDX6/MGLL/CDS2/INPP4A/SLC44A2/PCYT2/MTMR10/PLCB2/PLCG1/INPP5B	96
UCDvsCTRL.downregulated	UCDvsCTRL	downregulated	GO:0044242	cellular lipid catabolic process	71/1700	238/18986	0.2983193	3.331700	11.351840	0	0	0	PCK1/ACAT1/ACADM/LPIN2/APOB/ABHD6/ASAH2/ABHD2/HACL1/SORL1/SCARB1/CYP4F2/ECHDC2/ACOX1/APOA4/LCT/ENPP2/APOC3/HADHB/ACAA1/DDHD2/ENPP7/GALC/CYP4F12/BDH2/NEU1/CROT/CRAT/PCK2/GBA3/ABCD4/SMPD1/FUCA1/PHYH/PPARA/SLC27A4/AKR1B10/ETFDH/SIRT2/ACOX2/SMPD3/ACAA2/ABHD5/PRKCD/ABHD12/ASAH1/CYP4F3/SGPL1/ACADVL/ALDH3B1/PNPLA2/SCP2/ABCD1/SESN2/AIG1/PLCG2/AADAC/GPCPD1/NAGA/HADHA/LPIN3/ABCD3/PIK3CG/PEX5/CPT2/PNPLA6/PRDX6/MGLL/AUH/ACADS/PLCG1	71
UCDvsCTRL.downregulated	UCDvsCTRL	downregulated	GO:0016042	lipid catabolic process	89/1700	351/18986	0.2535613	2.831832	10.863179	0	0	0	PCK1/ACAT1/ACADM/CYP3A4/LPIN2/LIPA/APOB/ABHD6/ASAH2/ABHD2/HACL1/SORL1/SCARB1/CYP4F2/ECHDC2/ACOX1/APOA4/HSD17B11/NAAA/LCT/ENPP2/APOC3/HADHB/ACAA1/DPEP1/ABHD15/DDHD2/ENPP7/GALC/CYP4F12/BDH2/NEU1/CROT/CRAT/HPGD/PCK2/GBA3/PLCH2/ABCD4/SMPD1/FUCA1/PHYH/PPARA/SLC27A4/AKR1B10/ETFDH/SIRT2/CIDEC/ACOX2/SULT2A1/SMPD3/ACAA2/ABHD5/PLA2G12B/PRKCD/ABHD12/ASAH1/CYP4F3/SGPL1/ACADVL/ALDH3B1/PNPLA2/SCP2/PAFAH1B1/ABCD1/SESN2/THRA/AIG1/CYP27A1/PLCG2/AADAC/GPCPD1/NAGA/ABHD4/HADHA/SCT/LPIN3/CRTC3/ABCD3/PIK3CG/PEX5/CPT2/PNPLA6/PRDX6/MGLL/AUH/ACADS/PLCB2/PLCG1	89
UCDvsCTRL.downregulated	UCDvsCTRL	downregulated	GO:0044282	small molecule catabolic process	83/1700	375/18986	0.2213333	2.471903	9.028018	0	0	0	PCK1/ALDOB/ACAT1/ACADM/CYP3A4/LPIN2/SORD/ABHD2/HACL1/SCARB1/OAT/CYP4F2/ECHDC2/SULT1B1/PRODH/ACOX1/GDA/GK/HADHB/ACAA1/DPEP1/IDS/QPRT/CYP4F12/BDH2/CROT/CRAT/ENPP4/CRYL1/SLC25A44/SHMT1/PCK2/GLS/ACSS1/ABCD4/GNPDA1/HGD/PHYH/PPARA/SLC27A4/ACADSB/AKR1B10/SULT1A2/ETFDH/DERA/ACOX2/SULT2A1/ACAA2/CYP4F3/ADH4/ACADVL/ALDH3B1/ADA2/GK5/DLST/HAGH/ADA/SCP2/ABCD1/ENTPD7/ABAT/SESN2/IDNK/DPYD/AIG1/CYP27A1/ENTPD4/NAGK/HADHA/LPIN3/HMGCL/PPM1K/ABCD3/RBKS/PEX5/PGM1/SULT1A1/CPT2/GLUD1/AUH/ACADS/ALDH6A1/MGAT1	83
UCDvsCTRL.downregulated	UCDvsCTRL	downregulated	GO:0006639	acylglycerol metabolic process	44/1700	135/18986	0.3259259	3.640017	9.653593	0	0	0	PCK1/LPIN2/LIPA/APOB/ABHD6/ABHD2/SORL1/SCARB1/RGN/APOA4/DGAT2/DGKA/APOC3/GK/DGAT1/DDHD2/NR1H3/PCK2/G6PC1/GPAM/CIDEC/GPAT3/ABHD5/ABHD12/APOBEC1/MBOAT7/INSIG2/GK5/PNPLA2/LPGAT1/CAT/AVIL/MTTP/PGS1/CDK8/MOGAT2/AADAC/MOGAT3/LPIN3/NR1H2/PIK3CG/ACSL1/SREBF1/MGLL	44

You can visualize the results in many different ways, check the documentation of this package to see all options. For simplicity, we will plot only the dot plot here but explore other ways to visualize in your own analysis.

dotplot(ora_go) +
    default_theme() +
    theme(axis.text.x = element_text(angle=45, hjust = 1))

Gene Set Enrichment Analysis (GSEA)

Because GSEA uses the log2 fold-change to rank the genes, we first need to prepare this input vector with genes ordered based on their fold-change. In this case, we will not use compareCluster to do everything all at once, instead we will use a custom function to prepare the data and run the analysis. We don’t need the universe genes in this case.

# Define the function to create the ranked gene list and run the analysis
run_gsea <- function(data, contrast, database = c('GO-BP', 'KEGG', 'Reactome')) {

  # Creates the ordered gene list for given contrast
  foldchanges <- data$logFC[data$Contrast == contrast]
  names(foldchanges) <- data$entrezgene_id[data$Contrast == contrast]
  foldchanges <- sort(foldchanges, decreasing = TRUE)

  # Run the analysis for given database
  if(database == 'GO-BP') {
    res <- gseGO(geneList = foldchanges, OrgDb = "org.Hs.eg.db", keyType = "ENTREZID", ont = "BP", pvalueCutoff = 0.05)
  } else if(database == 'KEGG') {
    res <- gseKEGG(geneList = foldchanges, organism = 'hsa', pvalueCutoff = 0.05)
  } else if(database == 'Reactome') {
    res <- gsePathway(geneList = foldchanges, organism = 'human', pvalueCutoff = 0.05)
  } else {
    print("Choose a database among GO-BP, KEGG or Reactome.")
  }
  return(res)

}

Now apply the function to our contrasts. For simplicity, we will do only for GO-BP.

gsea_ucd_ctrl <- run_gsea(results_sig, contrast = 'UCDvsCTRL', database = 'GO-BP')

using 'fgsea' for GSEA analysis, please cite Korotkevich et al (2019).

preparing geneSet collections...

GSEA analysis...

leading edge analysis...

done...

gsea_ucd_tcd <- run_gsea(results_sig, contrast = 'UCDvsTCD', database = 'GO-BP')

using 'fgsea' for GSEA analysis, please cite Korotkevich et al (2019).

preparing geneSet collections...

GSEA analysis...

leading edge analysis...

done...

# Converts entrezid back to symbol
gsea_ucd_ctrl <- setReadable(gsea_ucd_ctrl, org.Hs.eg.db)
gsea_ucd_tcd <- setReadable(gsea_ucd_tcd, org.Hs.eg.db)

Similarly to ORA, we can check the results with @result. Note a column called NES, this is the Normalized Enrichment Score and the most important metric here to interpret the results. The NES is derived from the enrichment score (ES), a value that reflects the degree to which a gene set is overrepresented at the top (upregulated, positive ES) or bottom (downregulated, negative ES) of a ranked gene list. This score is calculated by a “random walk” algorithm that increases a running sum when it encounters a gene from the set and decreases it otherwise. The final ES is the maximum deviation from zero during this walk.

knitr::kable(head(gsea_ucd_ctrl))

	ID	Description	setSize	enrichmentScore	NES	pvalue	p.adjust	qvalue	rank	leading_edge	core_enrichment
GO:0051276	GO:0051276	chromosome organization	191	0.5468616	3.208765	0	0	0	984	tags=57%, list=26%, signal=44%	CDC20/TPX2/CCNB1/TOP2A/CENPE/KIF11/CENPF/NUSAP1/RACGAP1/HMGB3/HMGB2/REC8/MCM4/MYC/NDC1/SMC2/EZH2/TACC3/RCC1/SMC4/MCM3/PCNA/MCM5/KNTC1/KNL1/MCM7/CENPX/INCENP/RUVBL2/GNL3/HMGA1/NCAPD2/KIF22/CCT2/DKC1/RAN/ANAPC1/NASP/ERN2/PRKDC/NUP107/NCAPD3/MSH2/HSP90AA1/CCT5/LMNA/HSP90AB1/CCT3/MCM6/BANF1/NBN/PTGES3/TP53/HMGB1/SMARCC1/SSBP1/SMARCB1/NAT10/HNRNPC/HAT1/ACTL6A/NUP155/CCT7/RAD21/KPNB1/CCT6A/CDC27/BCCIP/XRCC6/TCP1/PARP1/CCT8/HNRNPA1/ITPA/XRCC5/CCT4/SMARCA4/PML/NOP10/HSPA1B/ATR/ACTB/HNRNPU/H3-3B/SUB1/KMT5A/PPP2R1A/DRG1/G3BP1/PHB2/STAG1/PBRM1/NUP133/RAB11A/NUDC/HNRNPA2B1/NUP62/MLH1/ASCC3/SMC1A/SEH1L/DHX9/DDX1/NAA50/HNRNPD/MCRS1/SMARCD2/CDK5RAP2/MCMBP
GO:0098813	GO:0098813	nuclear chromosome segregation	92	0.5938188	3.090306	0	0	0	265	tags=29%, list=7%, signal=28%	CDC20/TPX2/ASPM/CCNB1/TOP2A/CENPE/KIF11/CENPF/NUSAP1/AURKA/RACGAP1/ECT2/REC8/NDC1/SMC2/TACC3/RCC1/SMC4/KNTC1/KNL1/INCENP/NCAPD2/KIF22/RAN/ANAPC1/RCC2/NCAPD3
GO:0007059	GO:0007059	chromosome segregation	123	0.5627398	3.079081	0	0	0	265	tags=26%, list=7%, signal=25%	MKI67/CDC20/TPX2/ASPM/CCNB1/TOP2A/CENPE/KIF11/CENPF/NUSAP1/AURKA/RACGAP1/ECT2/REC8/GPSM2/NDC1/SMC2/TACC3/RCC1/SMC4/KNTC1/KNL1/CENPX/INCENP/NCAPD2/TUBB/KIF22/RAN/ANAPC1/RCC2/PLSCR1/NCAPD3
GO:0033044	GO:0033044	regulation of chromosome organization	93	0.5742037	2.987108	0	0	0	701	tags=51%, list=19%, signal=42%	CDC20/CCNB1/TOP2A/CENPE/CENPF/MYC/SMC2/TACC3/SMC4/KNTC1/KNL1/INCENP/RUVBL2/GNL3/NCAPD2/CCT2/DKC1/ANAPC1/NCAPD3/HSP90AA1/CCT5/LMNA/CCT3/NBN/PTGES3/TP53/SMARCC1/SMARCB1/NAT10/HNRNPC/ACTL6A/CCT7/RAD21/CCT6A/CDC27/TCP1/PARP1/CCT8/HNRNPA1/XRCC5/CCT4/SMARCA4/PML/ATR/ACTB/HNRNPU/H3-3B
GO:0000819	GO:0000819	sister chromatid segregation	72	0.5863184	2.923381	0	0	0	924	tags=58%, list=25%, signal=45%	CDC20/TPX2/CCNB1/TOP2A/CENPE/KIF11/CENPF/NUSAP1/RACGAP1/SMC2/TACC3/RCC1/SMC4/KNTC1/KNL1/INCENP/NCAPD2/KIF22/RAN/ANAPC1/NCAPD3/SMARCC1/SMARCB1/ACTL6A/RAD21/KPNB1/CDC27/BCCIP/SMARCA4/HSPA1B/ACTB/HNRNPU/KMT5A/PPP2R1A/DRG1/STAG1/PBRM1/RAB11A/NUDC/NUP62/SMC1A/SEH1L
GO:0051301	GO:0051301	cell division	179	0.4964854	2.902321	0	0	0	265	tags=23%, list=7%, signal=22%	CEP55/IQGAP3/CDC20/TPX2/ASPM/CCNB1/TOP2A/CENPE/KIF11/CENPF/CCNA2/NUSAP1/STMN1/AURKA/RACGAP1/ECT2/GPSM2/MYC/KIF20B/CDC25B/TIMELESS/SMC2/TACC3/CCND1/RCC1/SMC4/KNTC1/KNL1/CENPX/INCENP/CKAP2/GNL3/NCAPD2/TUBB/CKAP5/RAN/ANAPC1/RCC2/KAT2A/CDK2AP2/NCAPD3

Again, you can visualize the results in many ways. With the script below you can get an overview of the top 10 enriched pathways and the top 10 repressed pathways.

data <- gsea_ucd_ctrl
n <- 10

data_plot <- arrange(as.data.frame(data@result), desc(abs(NES))) %>%
    group_by(sign(NES)) %>%
    dplyr::slice(1:n)

ggplot(data_plot, showCategory = n*2,
      aes(NES, fct_reorder(Description,NES), fill = -log10(p.adjust))) +
geom_col() +
geom_vline(xintercept = 0, colour = '#696969', linetype = 'dashed') +
scale_fill_gradientn(colours=c("#b3eebe", "#46bac2", "#371ea3"),
                      guide=guide_colorbar(reverse=TRUE)) +
labs(x = 'Normalized Enrichment Score',
     y = '') +
default_theme() +
theme(text = element_text(size = 11))

Another interesting way of visualizing the GSEA results is the “running score” plot. For a given pathway, this plot shows the position of the genes in the ranked list and the enrichment score.

gseaplot(gsea_ucd_ctrl, geneSetID = 1, title = gsea_ucd_ctrl$Description[1])

Note the differences between the two methods, they won’t always give you the same results. Keep the differences between the two methods in mind when doing your analysis and interpreting the results.

Conclusion

Now that you are familiar with the basics of RNA-sequencing analysis, have fun analyzing the data you generated during the week in the lab :)